The invention relates generally to methods and systems for removing inherent autofluorescence and the contribution of dark current from images of biological materials.
Recent advances in imaging and microscopy technologies combined with the development of fluorescent probes have allowed researchers to study variations in movement, distribution, and concentration of various molecular markers. In many such studies, fluorescent probes with a binding specificity to one or more cellular molecules are introduced into a sample. Images are obtained of the sample, for example with fluorescence microscopy, and these images may be processed and further analyzed to determine the amount of fluorescence in the image, which may provide information about the cellular marker. Accurate detection and analysis of the contribution of fluorescence from the fluorescent probes may be critical for many microscopy applications, such as molecular pathology imaging. For example, the fluorescence in the image may be analyzed to determine if the samples contain markers associated with specific clinical conditions, such as cancer.
Cells in many organisms have inherent fluorescence, which may be referred to as autofluorescence. This autofluorescence can interfere with the analysis of images obtained of these cells. For example, autofluorescence may reduce signal detection sensitivity by masking the fluorescence of the probe of interest. In addition, autofluorescence may also decrease the specificity of detection by providing false positive results.
The extent of autofluorescence may be limited by treating the sample with certain dyes or chemicals prior to image acquisition. For example, the dye pontiamine sky blue is used to stain samples and quench autofluorescence. However, these treatment techniques involve further manipulation of the biological sample, which may degrade the quality of the sample itself. In addition, strategies for autofluorescence removal have been proposed that involve the image acquisition hardware, such as using lasers for excitation or polarized light. However, these techniques may not be suitable with standard fluorescent microscopes in widespread use.
Digitally acquired fluorescence microscope images can also be processed retrospectively using software methods, to separate autofluorescence from the relevant dye fluorescence. Some of these methods rely on acquiring estimates of the pure autofluorescence signal and using them to remove autofluorescence from images containing both dye and autofluorescence signals by a weighted subtraction. Others use statistical correlation techniques to correct for the additive autofluorescence signal. While these techniques may be more cost effective, they may not be able to completely remove the autofluorescence component from fluorescence microscopy images. For example, spectral unmixing methods often require prior knowledge of the amount of autofluorescence in the sample.
In addition, cell or tissue images may also include a certain amount of noise contributed by the acquisition system itself. For example, “dark current,” represents the response, in the absence of light, of many types of electromagnetic radiation receptors. Generally, dark current is dealt with by a simple subtraction. However, there is no image processing method that deals with both autofluorescence and dark current.